Inductive signaling plays a critical role in both normal and disease development as developmental pathways that become unregulated in the adult can lead to abnormal patterning, overproliferation and neoplasia. One signaling pathway that is involved in several patterning events during embryogenesis is that triggered by secreted sonic hedgehog (Shh) (Echelard, Y., Epstein, D. J., St-Jacques, B., Shen, L., Mohler, J., McMahon, J. A. & McMahon, A. P., Cell 75, 1417-1430 (1993); Riddle, R., Johnson, R. L., Laufer, E. & Tabin, C., Cell 75, 1401-1418 (1993); Krauss, S., Concordet, J.-P. & Ingham, P. W, Cell 75, 1431-1444 (1993); Roelink, H., Augsburger, A., Heemskerk, J., Korzh, V., Norlin, S., Ruiz i Altaba, A., Tanabe, Y., Placzek, M., Edlund, T., Jessell, T. M. & Dodd, J., Cell 76, 761-775 (1994)). Shh binding to the membrane patched (ptc)-smoothened (smo) receptor complex elicits a cascade of cytoplasmic signal transduction events, including the inhibition of protein kinase A (PKA) (Fan, C.-M., Porter, J. A., Chiang, C., Chang, D. T., Beachy, P. A. & Tessier-Lavigne, M., Cell 81, 457-465 (1995); Hynes, M., Porter, J. A., Chiang, C., Chang, D., Tessier-Lavigne, M., Beachy, P. A. & Rosenthal, A., Neuron 15, 35-34 (1995); Concordet, J.-P., Lewis, K. E., Moore, J., Goodrich, L. V., Johnson, R. L., Scott, M. P. & Ingham, P. W., Development 122, 2835-2846 (1996); Epstein, D. J., Martí, E., Scott, M. P. & McMahon, A. P., Development 122, 2885-2894 (1996); Goodrich, L. V., Johnson, R. L., Milenkovic, L., McMahon, J. A. & Scott, M. P., Genes Dev. 10, 301-312 (1996); Hammerschmidt, M., Bitgood, M. J. & McMahon, A. P., Genes and Dev. 10, 647-658 (1996); Marigo, V., Johnson, R. L., Vortkamp, A. & Tabin, C. J., Dev. Biol. 180, 273-283 (1996); Stone, D. M., Hynes, M., Armanini, M., Swanson, T. A., Gu, Q., Johnson, R. L., Scott, M. P., Pennica, D., Goddard, A., Phillips, H., Noll, M., Hooper, J. E., de Sauvage, F. & Rosenthal, A., Nature 384, 129-134 (1996)) that leads to the transcription of the zinc finger transcription factor gene Gli1 (Marigo, V., Johnson, R. L., Vortkamp, A. & Tabin, C. J., Dev. Biol. 180, 273-283 (1996); Lee, J., Platt, K. A., Censullo, P. & Ruiz i Altaba, A., Development (1997)). Gli1 is a proto-oncogene first isolated as an amplified gene in a glioma that can transform fibroblasts in cooperation with E1A (Kinzler, K. W., Bigner, S. H., Bigner, D. D., Trent, J. M., Law, M. L., O'Brien, S. J., Wong, A. J. & Vogelstin, B., Science 236, 70-73 (1987); Ruppert, J. M., Vogelstein, B. & Kinzler, K. W., Molecular and Cellular Biology 11, 1724-1728 (1991)). Gli1 is a member of a family comprising two other related genes: Gli2 and Gli3 (Ruppert, J. M., Vogelstein, B., Arheden, K. & Kinzler, K. W., Mol. Cell Biol. 10, 5408-5415 (1990); Hui, C.-C., Slusarski, D., Platt, K. A., Holmgren, R. & Joyner, A. L., Developmental Biology 162, 402-413 (1994)). However, only Gli1 has been shown to be a target of Shh and mimic its effects (Lee, J., Platt, K. A., Censullo, P. & Ruiz i Altaba, A., Development (1997)). In Drosophila, hedgehog signaling (Forbes, A. J., Nakano, Y., Taylor, A. M. & Ingham, P. W., Development Supplement 115-124 (1993)) similarly leads to the action of cubitus interruptus (ci), a Gli homolog that activates transcription of hedgehog-target genes (Domínguez, M., Brunner, M., Hafen, E. & Basler, K., Science 272, 1621-1625 (1996); Alexandre, C., Jacinto, A. & Ingham, P. W., Genes and Dev. 10, 2003-2013 (1996); Hepker, J., Wang, Q.-T., Motzny, C. K., Holmgren, R. & Orenic, T. V., Development 124, 549-558 (1997); von Ohnen, T., Lessing, D., Nusse, R. & Hooper, J. E., Proc. Natl. Acad. Sci. USA. 94, 2404-2409 (1997); Mullor, J. L., Calleja, M., Capdevila, J. & Guerrero, I., Development 124, 1227-1237 (1997)).
One of the processes in which Shh signaling is involved is the differentiation of ventral neural tube cell types acting as a notochord and floor plate-derived signal (Echelard, Y., Epstein, D. J., St-Jacques, B., Shen, L., Mohler, J., McMahon, J. A. & McMahon, A. P., Cell 75, 1417-1430 (1993); Roelink, H., Augsburger, A., Heemskerk, J., Korzh, V., Norlin, S., Ruiz i Altaba, A., Tanabe, Y., Placzek, M., Edlund, T., Jessell, T. M. & Dodd, J., Cell 76, 761-775 (1994); Martí, E., Bumcrot, D. A., Takada, R. & McMahon, A. P., Nature 375, 322-325 (1995); Ruiz i Altaba, A., Roelink, H. & Jessell, T. M., Mol. Cell. Neurosci. 6, 106-121 (1995); Chiang, C., Litingtung, Y., Lee, E., Young, K. E., Corden, J. L., Westphal, H. & Beachy, P. A., Nature 383, 407-413 (1996); Ericson, J., Morton, S., Kawakami, A., Roelink, H. & Jessell, T. M., Cell 87, 661-673 (1996)).
In addition to effects on neural tissue, it has been found that ectopic expression of Shh and Gli1 also leads to the activation of Shh signaling target genes in epidermal non-neural ectoderm. Injected Shh induced the ectopic expression of Gli1, HNF-3β and Shh (Ruiz i Altaba, A., Roelink, H. & Jessell, T. M., Mol. Cell. Neurosci. 6, 106-121 (1995)), and ectopic expression of Gli1 induced the ectopic expression of HNF-3β and Shh (Lee, J., Platt, K. A., Censullo, P. & Ruiz i Altaba, A. Gli1 is a target of sonic hedgehog that induces ventral neural tube development. Development (1997)). Together, these results indicated that both neural and epidermal cells have functional reception and transduction mechanisms for Shh and can respond by activating the expression of Shh/Gli1 target genes even though epidermal cells do not normally receive the Shh signal at this stage.
Furthermore, SHH signaling has been implicated in many aspects of animal development, acting through the transmembrane proteins PATCHED1 (PTCH1) and SMOH to activate the GLI zinc-finger transcription factors (Ingham, P. & McMahon, A., Genes Dev. 15, 3059-87 (2001); Ruiz i Altaba, A., Sanchez, P. & Dahmane, N., Nat. Rev. Cancer 2, 361-372 (2002).
A different signaling pathway is triggered by HEDGEHOG (HH) proteins, acting through the transmembrane proteins PATCHED1 (PTCH1) and SMOOTHENED (SMOH), to regulate the three GLI zinc finger transcription factors. SHH-GLI signaling is required for the growth of certain sporadic cancers, including basal cell carcinomas (BCCs) (Dahmane, N., et al., (1997). Activation of the transcription factor Gli1 and the Sonic hedgehog signalling pathway in skin tumours. Nature 389, 876-881; Williams et al., (2003) Identification of a small molecule inhibitor of the hedgehog signaling pathway: effects on basal cell carcinoma-like lesions. Proc Natl Acad Sci USA. 100, 4616-4621; Athar et al., (2004) Inhibition of smoothened signaling prevents ultraviolet B-induced basal cell carcinomas through regulation of Fas expression and apoptosis. Cancer Res. 64, 7545-7552, medulloblastomas (Dahmane et al., (2001) The Sonic Hedgehog-Gli pathway regulates dorsal brain growth and tumorigenesis. Development 128, 5201-5212; Berman et al., (2002) Medulloblastoma growth inhibition by hedgehog pathway blockade. Science 297, 1559-1561, gliomas (Dahmane et al., (2001) The Sonic Hedgehog-Gli pathway regulates dorsal brain growth and tumorigenesis. Development 128, 5201-5212, pancreatic (Thayer et al., (2003) Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis. Nature 425, 851-856, stomach (Berman et al., (2003) Widespread requirement for Hedgehog ligand stimulation in growth of digestive tract tumours. Nature 425, 846-851 and prostate cancers (Sanchez et al., (2004) Inhibition of prostate cancer proliferation by interference with SONIC HEDGEHOG-GLI1 signaling. Proc. Natl. Acad. Sci. USA. 101, 12561-12566; Karhadkar et al., (2004) Hedgehog signalling in prostate regeneration, neoplasia and metastasis. Nature 431, 707-712; Sheng et al., (2004) Sheng, T., Li, C., Zhang, X., Chi, S., He, N., Chen, K., McCormick, F., Gatalica, Z., Xie, J. (2004) Activation of the hedgehog pathway in advanced prostate cancer. Mol Cancer. 3, 29).
Alterations in several different oncogenic signaling pathways are found in many cancer types, begging the question of how such different events converge to produce reproducible and diagnosable tumors. An exception appears to be cutaneous melanomas in which activation of the RAS-RAF-MEK/AKT signaling is ubiquitous (Chin, (2003) The genetics of malignant melanoma: lessons from mouse and man. Nat Rev Cancer. 3, 559-570; Chudnovsky et al., (2005) Melanoma genetics and the development of rational therapeutics. J. Clin. Invest. 115, 813-824; Meier et al., (2005), The RAS/RAF/MEK/ERK and PI3K/AKT signaling pathways present molecular targets for the effective treatment of advanced melanoma. Front Biosci. 10, 2986-3001). Melanomas originate from melanocytes and/or from their neural crest-derived pluripotent precursors, and can develop, directly or from skin moles or nevi, into incurable distant metastasis in viscera, bone and brain.
These findings contrast with the fact that such tumors commonly harbor mutations in various oncogenes and tumor suppressors, including EGFR, RAS and PTEN (Vogelstein and Kinzler, (2004) Cancer genes and the pathways they control. Nat Med. 10, 789-799. It is not known whether the effects of such oncogenic mutations could affect the function of the GLI proteins. More particularly, it has yet to be determined whether SHH-GLI signaling could have a role in melanocytes and cutaneous melanoma. The findings of an association between the oncogenic mutations noted above and the function of the GLI proteins could address the need for novel therapeutics for the treatment of melanomas and other tumor types in which oncogenic mutations and the presence of GLI potentiates the tumorigenic process. The work presented herein addresses this need.
The molecular mechanisms underlying cancer development and metastases are beginning to be understood and with this the hope that rational and targeted cures can be developed. The incidence of cutaneous melanoma is increasing and it remains one of the deadliest cancers after metastasis. A large body of evidence gathered over many years shows that cutaneous human melanomas and their metastases routinely involve oncogenic activation of the RAS-RAF-MEK and AKT signaling pathways (Meier, F. et al. The RAS/RAF/MEK/ERK and PI3K/AKT signaling pathways present molecular targets for the effective treatment of advanced melanoma. Front Biosci. 10, 2986-3001 (2005)). Common alterations include those activating NRAS—which activates both the BRAF-MEK and AKT cascades—and BRAF; as well as increases in the level of AKT3, with AKT signaling being critical for melanomagenesis (Davies, H. et al. Mutations of the BRAF gene in human cancer. Nature 417, 949-954 (2002); Stahl, J. M. et al. Deregulated Akt3 activity promotes development of malignant melanoma. Cancer Res. 64, 7002-7010 (2004); Chudnovsky, Y., Khavari, P. A. & Adams, A. E. Melanoma genetics and the development of rational therapeutics. J. Clin. Invest. 115, 813-824 (2005)). In addition, we have recently shown that cutaneous melanomas depend on sustained SHH-GLI signaling—a cellular communication pathway involved in patterned growth and in stem cell/progenitor lineages—and on GLI1 function for growth, recurrence and metastasis (Stecca, B. et al. Melanocyte proliferation and melanoma growth, recurrence and metastasis require sustained SONIC HEDGEHOG-GLI signaling. Submitted. (2006)). These results raise the question of how signaling pathways involved in critical aspects of embryonic development, adult homeostasis and human cancers interact. Here we show that RAS-MEK/AKT and SHH signaling converge to regulate the behavior of GLI1, the function of which is required in human cancers, including prostate cancer and melanoma (Stecca, B. et al. Melanocyte proliferation and melanoma growth, recurrence and metastasis require sustained SONIC HEDGEHOG-GLI signaling. Submitted. (2006); Sanchez, P. et al. Inhibition of prostate cancer proliferation by interference with SONIC HEDGEHOG-GLI1 signaling. Proc. Natl. Acad. Sci. USA. 101, 12561-12566 (2004)). Moreover, we show that SUPPRESSOR OF FUSED (SUFUH) provides a critical counterbalance to oncogenic RAS-MEK/AKT and SHH pathway inputs.
The citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.